Nucleic acid molecule

ABSTRACT

The disclosure relates to a nucleic acid molecule isolated from a  Papaver somniferum  cultivar that produces the opiate alkaloid noscapine which comprises 10 genes involved in the biosynthesis of opiate alkaloids.

INTRODUCTION

This disclosure relates to the isolation and sequencing of a nucleicacid molecule that includes a gene cluster comprising 10 genes from anoscapine producing Papaver somniferum [opium poppy] cultivar;transgenic cells transformed with said nucleic acid molecule, sequencevariants of the genes; the use of said genes/proteins in the productionof opiate alkaloids; and the use of the genes as a marker of P.somniferum plants that synthesize opiate alkaloids, in particularnoscapine.

BACKGROUND TO DISCLOSURE

Noscapine belongs to the phthalideisoquinoline subclass of thestructurally diverse isoquinoline alkaloids whereas codeine, morphine,thebaine and oripavine belong to the morphinan subclass. While thebiosynthesis of morphinans has been elucidated at the molecular levelour knowledge of noscapine biosynthesis has not advanced significantlysince the demonstration using isotope labeling in the 1960s, that it isderived from scoulerine. Understanding the biochemical geneticsunderpinning noscapine biosynthesis should enable improved production ofnoscapine and related molecules both in poppy and other expressionsystems.

P. somniferum is the plant from which opium is extracted. The opiumpoppy is the only commercially exploited poppy of the familyPapaveraceae and is the principal source of natural opiates. The opiumis extracted from latex harvested from the green seed pods. A furthersource of opiate alkaloids is the poppy straw which is the dried matureplant. P. somniferum is a source of clinically useful opiate alkaloidssuch as morphine, codeine, thebaine, noscapine [also known as narcotine]and papaverine. The clinical application of these opiate alkaloids andtheir derivates is broad having use as analgesics, cough suppressantsand anti-spasmodics. Although not used as a pharmacological agent in itsown right, thebaine is a particularly useful opiate which can beconverted into a range of compounds such as hydrocodone, oxycodone,oxymorphone, nalbuphine naltrexone, buprenorphine and etorphine. Theseintermediates also have broad pharmaceutical applications. For example,oxycodone, oxymorphone and etorphine are widely used as an analgesic formoderate to severe pain and are often combined with other analgesicssuch as ibuprofen. Buprenorphine is used in the treatment of heroinaddiction and chronic pain. Naltrexone is used in the treatment ofalcohol and opiate addiction.

This disclosure relates to transcriptomic analysis of P. somniferumnoscapine producing cultivars compared to P. somniferum cultivars thatare non-noscapine producing. The analysis has revealed the exclusiveexpression of a group of mostly cytochrome P450 and methyltransferasegenes in a poppy variety that produces noscapine. These genes aresurprisingly absent from the genomes of two non-noscapine producingvarieties. Analysis of an F2 mapping population indicated the genes aretightly linked in the noscapine variety and bacterial artificialchromosome sequencing confirmed they exist as a novel gene cluster forthe biosynthesis of opiate alkaloids.

STATEMENTS OF INVENTION

According to an aspect of the invention there is provided an isolatednucleic acid molecule that encodes at least two polypeptides wherein thetwo polypeptides are selected from the group consisting of a nucleicacid molecule comprising or consisting of a nucleotide sequence selectedfrom:

-   -   i) a nucleotide sequence as represented by the sequence in SEQ        ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;    -   ii) a nucleotide sequence wherein said sequence is degenerate as        a result of the genetic code to the nucleotide sequence defined        in (i);    -   iii) a nucleic acid molecule the complementary strand of which        hybridizes under stringent hybridization conditions to the        sequence in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 wherein        said nucleic acid molecule encodes polypeptides involved in the        biosynthesis of P. somniferum opiate alkaloids or intermediates        in the biosynthesis of opiate alkaloids;    -   iv) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence as represented in SEQ ID NO: 11, 12, 13,        14, 15, 16, 17, 18, 19 or 20;    -   v) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence wherein said amino acid sequence is        modified by addition deletion or substitution of at least one        amino acid residue as represented in iv) above and which has        retained or enhanced opiate alkaloid biosynthetic activity.

According to a further aspect of the invention there is provided anisolated nucleic acid molecule that comprises a gene cluster thatencodes two or more polypeptides involved in the biosynthesis of opiatealkaloids or intermediates, wherein one of said two genes comprises anucleotide sequence selected from the group consisting of:

-   -   i) a nucleotide sequence as set forth in SEQ ID NO: 8;    -   ii) a nucleotide sequence wherein said sequence is degenerate as        a result of the genetic code to the nucleotide sequence defined        in (i);    -   iii) a nucleic acid molecule the complementary strand of which        hybridizes under stringent hybridization conditions to the        nucleotide sequence in SEQ ID NO: 8 and which encodes a        polypeptide that has carboxylesterase activity; and    -   iv) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence as set forth in SEQ ID NO: 18 or a        nucleotide sequence that encodes a polypeptide that has 46%        amino acid sequence identity across the full length amino acid        sequence set forth in SEQ ID NO: 18 wherein said polypeptide has        carboxylesterase activity.

According to a further aspect or embodiment of the invention there isprovided an isolated nucleic acid molecule that comprises a gene clusterthat encodes two or more polypeptides involved in the biosynthesis ofopiate alkaloids or intermediates, wherein one of said two genescomprises a nucleotide sequence selected from the group consisting of;

-   -   i) a nucleotide sequence as set forth in SEQ ID NO: 9;    -   ii) a nucleotide sequence wherein said sequence is degenerate as        a result of the genetic code to the nucleotide sequence defined        in (i);    -   iii) a nucleic acid molecule the complementary strand of which        hybridizes under stringent hybridization conditions to the        sequence in SEQ ID NO: 9 and which encodes a polypeptide that        has short-chain dehydrogenase/reductase activity; and    -   iv) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence as set forth in SEQ ID NO: 19 or a        nucleotide sequence that encodes a polypeptide that has is 46%        amino acid sequence identity across the full length amino acid        sequence set forth in SEQ ID NO: 19 wherein said polypeptide has        short-chain dehydrogenase/reductase activity.

Hybridization of a nucleic acid molecule occurs when two complementarynucleic acid molecules undergo an amount of hydrogen bonding to eachother. The stringency of hybridization can vary according to theenvironmental conditions surrounding the nucleic acids, the nature ofthe hybridization method, and the composition and length of the nucleicacid molecules used. Calculations regarding hybridization conditionsrequired for attaining particular degrees of stringency are discussed inSambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001); and Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes Part I, Chapter 2(Elsevier, New York, 1993). The T_(m) is the temperature at which 50% ofa given strand of a nucleic acid molecule is hybridized to itscomplementary strand. The following is an exemplary set of hybridizationconditions and is not limiting:

Very High Stringency (Allows Sequences that Share at Least 90% Identityto Hybridize)

-   -   Hybridization: 5×SSC at 65° C. for 16 hours    -   Wash twice: 2×SSC at room temperature (RT) for 15 minutes each    -   Wash twice: 0.5×SSC at 65° C. for 20 minutes each        High Stringency (Allows Sequences that Share at Least 80%        Identity to Hybridize)    -   Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours    -   Wash twice: 2×SSC at RT for 5-20 minutes each    -   Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each        Low Stringency (Allows Sequences that Share at Least 50%        Identity to Hybridize)    -   Hybridization: 6×SSC at RT to 55° C. for 16-20 hours    -   Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes        each.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleotide sequence as represented SEQ ID NO:1 wherein said nucleic acid molecule encodes a polypeptide with methyltransferase activity.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleotide sequence as represented SEQ ID NO:2 wherein said nucleic acid molecule encodes a polypeptide with methyltransferase activity.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleotide sequence as represented SEQ ID NO:3 wherein said nucleic acid molecule encodes a polypeptide with methyltransferase activity.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleotide sequence as represented SEQ ID NO:4 wherein said nucleic acid molecule encodes a polypeptide withcytochrome P450 activity.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleotide sequence as represented SEQ ID NO:5 wherein said nucleic acid molecule encodes a polypeptide withcytochrome P450 activity.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleotide sequence as represented SEQ ID NO:6 wherein said nucleic acid molecule encodes a polypeptide withcytochrome P450 activity.

In a preferred aspect or embodiment of the invention said nucleic acidmolecule comprises or consists of a nucleotide sequence as representedSEQ ID NO: 7 wherein said nucleic acid molecule encodes a polypeptidewith cytochrome P450 activity.

In a preferred aspect or embodiment of the invention said nucleic acidmolecule comprises or consists of a nucleotide sequence as representedSEQ ID NO: 8 wherein said nucleic acid molecule encodes a polypeptidewith carboxylesterase activity.

In a preferred aspect or embodiment of the invention said nucleic acidmolecule comprises or consists of a nucleotide sequence as representedSEQ ID NO: 9 wherein said nucleic acid molecule encodes a polypeptidewith short-chain dehydrogenase/reductase activity.

In a preferred aspect or embodiment of the invention said nucleic acidmolecule comprises or consists of a nucleotide sequence as representedSEQ ID NO: 10 wherein said nucleic acid molecule encodes a polypeptidewith acetyltransferase activity.

In a preferred embodiment of the invention said nucleic acid moleculeincludes SEQ ID NO: 1 and further includes one or more nucleotidesequences selected from the group consisting of: SEQ ID NO: 2, 3, 4, 5,6, 7, 8, 9 or 10.

In a preferred embodiment of the invention said nucleic acid moleculeincludes 3, 4, 5, 6, 7, 8 or 9 nucleotide sequences selected from thegroup consisting of: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In a preferred embodiment of the invention said nucleic acid moleculeincludes each of the nucleotide sequences as represented in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

According to a further aspect of the invention there is provided anisolated polypeptide selected from the group consisting of:

-   -   i) a polypeptide comprising or consisting of an amino acid        sequence as represented in SEQ ID NO: 17; or    -   ii) a modified polypeptide comprising or consisting of a        modified amino acid sequence wherein said polypeptide is        modified by addition deletion or substitution of at least one        amino acid residue of the sequence presented in SEQ ID NO: 17        and which has retained or enhanced cytochrome P450 activity.

In a preferred embodiment of the invention said polypeptide comprises orconsists of an amino acid sequence that is at least 55% identical to thefull length amino acid sequence in SEQ ID NO: 17 and which encodes apolypeptide with cytochrome P450 activity.

According to a further aspect of the invention there is provided anisolated polypeptide selected from the group consisting of:

-   -   i) a polypeptide comprising or consisting of an amino acid        sequence as represented in SEQ ID NO:18; or    -   ii) a modified polypeptide comprising or consisting of a        modified amino acid sequence wherein said polypeptide is        modified by addition deletion or substitution of at least one        amino acid residue of the sequence presented in SEQ ID NO: 18        and which has retained or enhanced carboxylesterase activity.

In a preferred embodiment of the invention said polypeptide comprises orconsists of an amino acid sequence that is at least 46% identical to thefull length amino acid sequence in SEQ ID NO: 18 and which encodes apolypeptide with carboxylesterase activity.

According to a further aspect of the invention there is provided anisolated polypeptide selected from the group consisting of:

-   -   i) a polypeptide comprising or consisting of an amino acid        sequence as represented in SEQ ID NO: 19; or    -   ii) a modified polypeptide comprising or consisting of a        modified amino acid sequence wherein said polypeptide is        modified by addition deletion or substitution of at least one        amino acid residue of the sequence presented in SEQ ID NO: 19        and which has retained or enhanced short-chain        dehydrogenase/reductase activity.

In a preferred embodiment of the invention said polypeptide comprises orconsists of an amino acid sequence that is at least 47% identical to thefull length amino acid sequence in SEQ ID NO: 19 and which encodes apolypeptide with short-chain dehydrogenase/reductase activity.

According to a further aspect of the invention there is provided anisolated polypeptide selected from the group consisting of:

-   -   i) a polypeptide comprising or consisting of an amino acid        sequence as represented in SEQ ID NO: 20; or    -   ii) a modified polypeptide comprising or consisting of a        modified amino acid sequence wherein said polypeptide is        modified by addition deletion or substitution of at least one        amino acid residue of the sequence presented in SEQ ID NO: 20        and which has retained or enhanced acetyltransferase activity.

In a preferred embodiment of the invention said polypeptide comprises orconsists of an amino acid sequence that is at least 67% identical to thefull length amino acid sequence in SEQ ID NO: 20 and which encodes apolypeptide with acetyltransferase activity.

A modified polypeptide as herein disclosed may differ in amino acidsequence by one or more substitutions, additions, deletions, truncationsthat may be present in any combination. Among preferred variants arethose that vary from a reference polypeptide by conservative amino acidsubstitutions. Such substitutions are those that substitute a givenamino acid by another amino acid of like characteristics. The followingnon-limiting list of amino acids are considered conservativereplacements (similar): a) alanine, serine, and threonine; b) glutamicacid and aspartic acid; c) asparagine and glutamine d) arginine andlysine; e) isoleucine, leucine, methionine and valine and f)phenylalanine, tyrosine and tryptophan. Most highly preferred arevariants that retain or enhance the same biological function andactivity as the reference polypeptide from which it varies.

In one embodiment, the variant polypeptides have at least 39% to 50%identity, even more preferably at least 55% identity, still morepreferably at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% identity, andat least 99% identity with most or the full length amino acid sequenceillustrated herein.

According to an aspect of the invention there is provided an isolatednucleic acid molecule comprising or consisting of a nucleotide sequenceselected from the group consisting of:

-   -   i) a nucleotide sequence as represented by the sequence in SEQ        ID NO: 7, 8, 9 or 10;    -   ii) a nucleotide sequence wherein said sequence is degenerate as        a result of the genetic code to the nucleotide sequence defined        in (i);    -   iii) a nucleic acid molecule the complementary strand of which        hybridizes under stringent hybridization conditions to the        sequence in SEQ ID NO: 7, 8, 9 or 10 wherein said nucleic acid        molecule encodes polypeptides involved in the biosynthesis of P.        somniferum opiate alkaloids or intermediates in the biosynthesis        of opiate alkaloids;    -   iv) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence as represented in SEQ ID NO: 17, 18, 19        or 20;    -   v) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence wherein said amino acid sequence is        modified by addition deletion or substitution of at least one        amino acid residue as represented in iv) above and which has        retained or enhanced opiate alkaloid biosynthetic activity.

According to a further aspect of the invention there is provided avector comprising a nucleic acid molecule according to the invention.

Preferably the nucleic acid molecule in the vector is under the controlof, and operably linked to, an appropriate promoter or other regulatoryelements for transcription in a host cell such as a microbial, (e.g.bacterial, yeast), or plant cell. The vector may be a bi-functionalexpression vector which functions in multiple hosts. In the case ofgenomic DNA this may contain its own promoter or other regulatoryelements and in the case of cDNA this may be under the control of anappropriate promoter or other regulatory elements for expression in thehost cell.

By “promoter” is meant a nucleotide sequence upstream from thetranscriptional initiation site and which contains all the regulatoryregions required for transcription. Suitable promoters includeconstitutive, tissue-specific, inducible, developmental or otherpromoters for expression in plant cells comprised in plants depending ondesign. Such promoters include viral, fungal, bacterial, animal andplant-derived promoters capable of functioning in plant cells.

Constitutive promoters include, for example CaMV 35S promoter (Odell etal. (1985) Nature 313, 9810-812); rice actin (McElroy et al. (1990)Plant Cell 2: 163-171); ubiquitin (Christian et al. (1989) Plant Mol.Biol. 18 (675-689); pEMU (Last et al. (1991) Theor Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3. 2723-2730); ALS promoter(U.S. application Ser. No. 08/409,297), and the like. Other constitutivepromoters include those in U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680, 5,268,463; and 5,608,142,each of which is incorporated by reference.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducedgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 andMcNellis et al. (1998) Plant J. 14(2): 247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156, herein incorporated by reference.

Where enhanced expression in particular tissues is desired,tissue-specific promoters can be utilised. Tissue-specific promotersinclude those described by Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792-803;Hansen et al. (1997) Mol. Gen. Genet. 254(3): 337-343; Russell et al.(1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) PlantPhysiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol.112(2): 525-535; Canevascni et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5): 773-778; Lam(1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993)Plant Mol. Biol. 23(6): 1129-1138; Mutsuoka et al. (1993) Proc. Natl.Acad. Sci. USA 90 (20): 9586-9590; and Guevara-Garcia et al (1993) PlantJ. 4(3): 495-50.

“Operably linked” means joined as part of the same nucleic acidmolecule, suitably positioned and oriented for transcription to beinitiated from the promoter. DNA operably linked to a promoter is “undertranscriptional initiation regulation” of the promoter. In a preferredaspect, the promoter is a tissue specific promoter, an induciblepromoter or a developmentally regulated promoter.

Particular of interest in the present context are nucleic acidconstructs which operate as plant vectors. Specific procedures andvectors previously used with wide success in plants are described byGuerineau and Mullineaux (1993) (Plant transformation and expressionvectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOSScientific Publishers, pp 121-148. Suitable vectors may include plantviral-derived vectors (see e.g. EP194809). If desired, selectablegenetic markers may be included in the construct, such as those thatconfer selectable phenotypes such as resistance to herbicides (e.g.kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate,gentamycin, spectinomycin, imidazolinones and glyphosate).

In a preferred embodiment of the invention said vector is a bacterialartificial chromosome [BACS].

According to a further aspect of the invention there is provided atransgenic cell transformed or transfected with a nucleic acid moleculeor vector according to the invention.

In a preferred embodiment of the invention said cell is a plant cell.

In a preferred embodiment of the invention said plant cell is from thegenus Papaver.

In a preferred embodiment of the invention said plant cell is a Papaversomniferum cell.

According to a further aspect of the invention there is provided a plantcomprising a plant cell according to the invention.

In a preferred embodiment of the invention said plant is from the genusPapaver; preferably Papaver somniferum.

In an alternative preferred embodiment of the invention said cell is amicrobial cell; preferably a bacterial or fungal cell [e.g. yeast,Saccharomyces cerevisae].

In a preferred embodiment of the invention said cell is adapted suchthat the nucleic acid molecule encoding one or more polypeptidesaccording to the invention is over-expressed when compared to anon-transgenic cell of the same species.

According to a further aspect of the invention there is provided anucleic acid molecule comprising a transcription cassette wherein saidcassette includes a nucleotide sequence designed with reference to anucleotide sequence selected from the group: SEQ ID NO: 1, 2, 3, 4, 5,6, 7, 8, 9 or 10, and is adapted for expression by provision of at leastone promoter operably linked to said nucleotide sequence such that bothsense and antisense molecules are transcribed from said cassette.

In a preferred embodiment of the invention said cassette is adapted suchthat both sense and antisense ribonucleic acid molecules are transcribedfrom said cassette wherein said sense and antisense nucleic acidmolecules are adapted to anneal over at least part or all of theirlength to form a inhibitory RNA or short hairpin RNA.

In a preferred embodiment of the invention said cassette is providedwith at least two promoters adapted to transcribe both sense andantisense strands of said ribonucleic acid molecule.

In an alternative preferred embodiment of the invention said cassettecomprises a nucleic acid molecule wherein said molecule comprises afirst part linked to a second part wherein said first and second partsare complementary over at least part of their sequence and furtherwherein transcription of said nucleic acid molecule produces anribonucleic acid molecule which forms a double stranded region bycomplementary base pairing of said first and second parts therebyforming an short hairpin RNA.

A technique to specifically ablate gene function is through theintroduction of double stranded RNA, also referred to as smallinhibitory/interfering RNA (siRNA) or short hairpin RNA [shRNA], into acell which results in the destruction of mRNA complementary to thesequence included in the siRNA/shRNA molecule. The siRNA moleculecomprises two complementary strands of RNA (a sense strand and anantisense strand) annealed to each other to form a double stranded RNAmolecule. The siRNA molecule is typically derived from exons of the genewhich is to be ablated. The mechanism of RNA interference is beingelucidated. Many organisms respond to the presence of double strandedRNA by activating a cascade that leads to the formation of siRNA. Thepresence of double stranded RNA activates a protein complex comprisingRNase III which processes the double stranded RNA into smaller fragments(siRNAs, approximately 21-29 nucleotides in length) which become part ofa ribonucleoprotein complex. The siRNA acts as a guide for the RNasecomplex to cleave mRNA complementary to the antisense strand of thesiRNA thereby resulting in destruction of the mRNA.

In a preferred embodiment of the invention said nucleic acid molecule ispart of a vector adapted for expression in a plant cell.

According to a further aspect of the invention there is provided a plantcell transfected with a nucleic acid molecule or vector according to theinvention wherein said cell has reduced expression of a polypeptideaccording to the invention.

According to an aspect of the invention there is provided a process forthe modification of one or more opiate alkaloids comprising:

-   -   i) providing a transgenic plant cell according to the invention;    -   ii) cultivating said plant cell to produce a transgenic plant;        and optionally    -   i) harvesting said transgenic plant, or part thereof.

In a preferred method of the invention said harvested plant material isdried and opiate alkaloid is extracted.

According to an alternative aspect of the invention there is provided aprocess for the modification of one or more opiate alkaloids or opiatealkaloid intermediate metabolites comprising:

-   -   i) providing a transgenic microbial cell according to the        invention that expresses one or more nucleic acid molecules        according to the invention in culture with at least one opiate        alkaloid or opiate alkaloid intermediate metabolite;    -   ii) cultivating the microbial cell under conditions that modify        one or more opitate alkaloid or opiate alkaloid intermediate;        and optionally    -   iii) isolating said opiate alkaloid or opiate alkaloid        intermediate from the microbial cell or cell culture.

In a preferred method of the invention said microbial cell is abacterial cell or fungal/yeast cell.

If microbial cells are used as organisms in the process according to theinvention they are grown or cultured in the manner with which theskilled worker is familiar, depending on the host organism. As a rule,microorganisms are grown in a liquid medium comprising a carbon source,usually in the form of sugars, a nitrogen source, usually in the form oforganic nitrogen sources such as yeast extract or salts such as ammoniumsulfate, trace elements such as salts of iron, manganese and magnesiumand, if appropriate, vitamins, at temperatures of between 0° C. and 100°C., preferably between 10° C. and 60° C., while gassing in oxygen.

The pH of the liquid medium can either be kept constant, that is to sayregulated during the culturing period, or not. The cultures can be grownbatchwise, semi-batchwise or continuously. Nutrients can be provided atthe beginning of the fermentation or fed in semi-continuously orcontinuously. The methylated opiate alkaloids produced can be isolatedfrom the organisms as described above by processes known to the skilledworker, for example by extraction, distillation, crystallization, ifappropriate precipitation with salt, and/or chromatography. To this end,the organisms can advantageously be disrupted beforehand. In thisprocess, the pH value is advantageously kept between pH 4 and 12,preferably between pH 6 and 9, especially preferably between pH 7 and 8.

The culture medium to be used must suitably meet the requirements of thestrains in question. Descriptions of culture media for variousmicroorganisms can be found in the textbook “Manual of Methods forGeneral Bacteriology” of the American Society for Bacteriology(Washington D.C., USA, 1981).

As described above, these media which can be employed in accordance withthe invention usually comprise one or more carbon sources, nitrogensources, inorganic salts, vitamins and/or trace elements.

Preferred carbon sources are sugars, such as mono-, di- orpolysaccharides. Examples of carbon sources are glucose, fructose,mannose, galactose, ribose, sorbose, ribulose, lactose, maltose,sucrose, raffinose, starch or cellulose. Sugars can also be added to themedia via complex compounds such as molasses or other by-products fromsugar refining. The addition of mixtures of a variety of carbon sourcesmay also be advantageous. Other possible carbon sources are oils andfats such as, for example, soya oil, sunflower oil, peanut oil and/orcoconut fat, fatty acids such as, for example, palmitic acid, stearicacid and/or linoleic acid, alcohols and/or polyalcohols such as, forexample, glycerol, methanol and/or ethanol, and/or organic acids suchas, for example, acetic acid and/or lactic acid.

Nitrogen sources are usually organic or inorganic nitrogen compounds ormaterials comprising these compounds. Examples of nitrogen sourcescomprise ammonia in liquid or gaseous form or ammonium salts such asammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate or ammonium nitrate, nitrates, urea, amino acids or complexnitrogen sources such as cornsteep liquor, soya meal, soya protein,yeast extract, meat extract and others. The nitrogen sources can be usedindividually or as a mixture.

Inorganic salt compounds which may be present in the media comprise thechloride, phosphorus and sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

Inorganic sulfur-containing compounds such as, for example, sulfates,sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or elseorganic sulfur compounds such as mercaptans and thiols may be used assources of sulfur for the production of sulfur-containing finechemicals, in particular of methionine.

Phosphoric acid, potassium dihydrogenphosphate or dipotassiumhydrogenphosphate or the corresponding sodium-containing salts may beused as sources of phosphorus.

Chelating agents may be added to the medium in order to keep the metalions in solution. Particularly suitable chelating agents comprisedihydroxyphenols such as catechol or protocatechuate and organic acidssuch as citric acid.

The fermentation media used according to the invention for culturingmicroorganisms usually also comprise other growth factors such asvitamins or growth promoters, which include, for example, biotin,riboflavin, thiamine, folic acid, nicotinic acid, panthothenate andpyridoxine. Growth factors and salts are frequently derived from complexmedia components such as yeast extract, molasses, cornsteep liquor andthe like. It is moreover possible to add suitable precursors to theculture medium. The exact composition of the media compounds heavilydepends on the particular experiment and is decided upon individuallyfor each specific case. Information on the optimization of media can befound in the textbook “Applied Microbiol. Physiology, A PracticalApproach” (Editors P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp.53-73, ISBN 0 19 963577 3). Growth media can also be obtained fromcommercial suppliers, for example Standard 1 (Merck) or BHI (brain heartinfusion, DIFCO) and the like.

All media components are sterilized, either by heat (20 min at 1.5 barand 121° C.) or by filter sterilization. The components may besterilized either together or, if required, separately. All mediacomponents may be present at the start of the cultivation or addedcontinuously or batchwise, as desired.

The culture temperature is normally between 15° C. and 45° C.,preferably at from 25° C. to 40° C., and may be kept constant or may bealtered during the experiment. The pH of the medium should be in therange from 5 to 8.5, preferably around 7.0. The pH for cultivation canbe controlled during cultivation by adding basic compounds such assodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia oracidic compounds such as phosphoric acid or sulfuric acid. Foaming canbe controlled by employing antifoams such as, for example, fatty acidpolyglycol esters. To maintain the stability of plasmids it is possibleto add to the medium suitable substances having a selective effect, forexample antibiotics. Aerobic conditions are maintained by introducingoxygen or oxygen-containing gas mixtures such as, for example, ambientair into the culture. The temperature of the culture is normally 20° C.to 45° C. and preferably 25° C. to 40° C. The culture is continued untilformation of the desired product is at a maximum. This aim is normallyachieved within 10 to 160 hours.

The fermentation broth can then be processed further. The biomass may,according to requirement, be removed completely or partially from thefermentation broth by separation methods such as, for example,centrifugation, filtration, decanting or a combination of these methodsor be left completely in said broth. It is advantageous to process thebiomass after its separation.

However, the fermentation broth can also be thickened or concentratedwithout separating the cells, using known methods such as, for example,with the aid of a rotary evaporator, thin-film evaporator, falling-filmevaporator, by reverse osmosis or by nanofiltration. Finally, thisconcentrated fermentation broth can be processed to obtain the opiatealkaloids present therein.

According to a further aspect of the invention there is provided the useof a gene encoded by a nucleic acid molecule as represented by thenucleic acid sequence in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, ora nucleic acid molecule that hybridizes under stringent hybridizationconditions to the nucleotide sequence in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 and encodes a polypeptide with opiate alkaloid biosyntheticactivity as a means to identify a locus wherein said locus is associatedwith altered expression or activity of said opiate alkaloid biosyntheticactivity.

Mutagenesis as a means to induce phenotypic changes in organisms is wellknown in the art and includes but is not limited to the use of mutagenicagents such as chemical mutagens [e.g. base analogues, deaminatingagents, DNA intercalating agents, alkylating agents, transposons,bromine, sodium azide] and physical mutagens [e.g. ionizing radiation,psoralen exposure combined with UV irradiation].

According to a further aspect of the invention there is provided amethod to produce a P. somniferum plant that has altered expression of apolypeptide according to the invention comprising the steps of:

-   -   i) mutagenesis of wild-type seed from a P. somniferum plant that        does express said polypeptide;    -   ii) cultivation of the seed in i) to produce first and        subsequent generations of plants;    -   iii) obtaining seed from the first generation plant and        subsequent generations of plants;    -   iv) determining if the seed from said first and subsequent        generations of plants has altered nucleotide sequence and/or        altered expression of said polypeptide;    -   v) obtaining a sample and analysing the nucleic acid sequence of        a nucleic acid molecule selected from the group consisting of:        -   a) a nucleic acid molecule comprising a nucleotide sequence            as represented in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;        -   b) a nucleic acid molecule that hybridises to the nucleic            acid molecule in a) under stringent hybridisation conditions            and that encodes a polypeptide with opiate alkaloid            biosynthsynthetic activity; and optionally    -   vi) comparing the nucleotide sequence of the nucleic acid        molecule in said sample to a nucleotide sequence of a nucleic        acid molecule of the original wild-type plant.

In a preferred method of the invention said nucleic acid molecule isanalysed by a method comprising the steps of:

-   -   i) extracting nucleic acid from said mutated plants;    -   ii) amplification of a part of said nucleic acid molecule by a        polymerase chain reaction;    -   iii) forming a preparation comprising the amplified nucleic acid        and nucleic acid extracted from wild-type seed to form        heteroduplex nucleic acid;    -   iv) incubating said preparation with a single stranded nuclease        that cuts at a region of heteroduplex nucleic acid to identify        the mismatch in said heteroduplex; and    -   v) determining the site of the mismatch in said nucleic acid        heteroduplex.

In a preferred method of the invention said P. somniferum plant hasenhanced opiate alkaloid biosynthetic activity.

In an alternative preferred method of the invention said P. somniferumplant has reduced or abrogated opiate alkaloid biosynthetic activity.

According to a further aspect of the invention there is provided a P.somniferum plant obtained by the method according to the invention.

According to an aspect of the invention there is provided a P.somniferum plant wherein said plant comprises a viral vector thatincludes all or part of a gene comprising a nucleic acid moleculeaccording to the invention.

In a preferred embodiment of the invention said gene or part is encodedby a nucleic acid molecule comprising a nucleic acid sequence selectedfrom the group consisting of:

-   -   i) a nucleic acid molecule comprising a nucleotide sequence as        represented in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;    -   ii) a nucleic acid molecule comprising a nucleotide sequence        that hybridises under stringent hybridisation conditions to a        nucleic acid molecule in (i) and which encodes a polypeptide        opiate alkaloid biosynthetic activity.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleotide sequence as represented in SEQ IDNO: 21.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleotide sequence as represented in SEQ IDNO: 22.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleotide sequence as represented in SEQ IDNO: 23.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleotide sequence as represented in SEQ IDNO: 24.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleotide sequence as represented in SEQ IDNO: 25.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleotide sequence as represented in SEQ IDNO: 26.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleotide sequence as represented in SEQ IDNO: 27.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleotide sequence as represented in SEQ IDNO: 28.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleotide sequence as represented in SEQ IDNO: 29.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleotide sequence as represented in SEQ IDNO: 30.

According to a further aspect of the invention there is provided a viralvector comprising all or part of a nucleic acid molecule according tothe invention.

According to an aspect of the invention there is provided the use of aviral vector according to the invention in viral induced gene silencingin a P. somniferum plant.

Virus induced gene silencing [VIGS] is known in the art and exploits aRNA mediated antiviral defence mechanism. Plants that are infected withan unmodified virus induces a mechanism that specifically targets theviral genome. However, viral vectors which are engineered to includenucleic acid molecules derived from host plant genes also inducespecific inhibition of viral vector expression and additionally targethost mRNA. This allows gene specific gene silencing without geneticmodification of the plant genome and is essentially a non-transgenicmodification.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, means “including but not limited to”, andis not intended to (and does not) exclude other moieties, additives,components, integers or steps. “Consisting essentially” means having theessential integers but including integers which do not materially affectthe function of the essential integers.

Throughout the description and claims of this specification, thesingular encompasses the plural unless the context otherwise requires.In particular, where the indefinite article is used, the specificationis to be understood as contemplating plurality as well as singularity,unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith.

An embodiment of the invention will now be described by example only andwith reference to the following figures:

FIG. 1: Identification of genes exclusively present in the genome of anoscapine producing poppy variety, HN1 (High Noscapine 1). (A) Relativeabundance of the major alkaloids extracted from the capsules of threecommercial varieties of poppy, HM1 (High Morphine 1), HT1 (HighThebaine 1) and HN1. M=morphine, C=codeine, T=thebaine, O=oripavine andN=Noscapine. (B) EST libraries from stem and capsule were generated bypyrosequencing and unique contiguous sequences assembled as described inmaterial and methods. Ten genes (PSMT1, PSMT2, PSMT3, CYP82X1, CYP82X2,CYP82Y1, CYP719A21, PSAT1, PSSDR1 and PSCXE1) as defined in the text,were represented only in EST libraries from the HN1 variety. ESTabundance of five other functionally characterized P. somniferum genes(BBE, TNMT, SalR, SalAT and T6DM) show them to be expressed in all threevarieties and at consistently higher levels in stem compared to capsuleas is also the case for the HN1 specific genes as shown in colour code(FIG. 1). PCR on genomic DNA from all three varieties revealed that theten HN1 specific genes are absent from the genomes of the HM1 and HT1varieties (FIG. 5);

FIG. 2: Segregation analysis of noscapine content in an F2 mappingpopulation demonstrates requirement for the noscapine gene cluster. (A)Box plot depiction of noscapine levels as percentage dry weight (DW) inglasshouse grown parental lines HN1 and HM1 and the F1 generation. (B)The field grown F2 generation segregated into three classes of zero, lowand high noscapine. F2 GC− and F2 GC+ indicate the absence and presencerespectively of the noscapine gene cluster. Numbers in brackets indicatenumber of individuals in each class;

FIG. 3: The HN1 gene cluster. The structure and position of the ten HN1specific genes expressed in stems and capsule tissues is shown above thecentral black line which represents 401 Kb of genomic sequence. Exonsare represented by filled grey boxes and introns by fine black lines.Arrows indicate the 5′ to 3′ orientation of each gene. Additional openreading frames depicted below the central black line are as defined bythe key. None of these ORFs are represented in the stem and capsule ESTlibraries;

FIG. 4: Functional characterisation using virus induced gene silencingof 6 genes from the HN1 gene cluster. Results from both leaf latex andcapsules are consistent with each of these genes encoding enzymesinvolved in noscapine biosynthesis (A-F). All compounds that accumulate,apart from scoulerine, have been putatively identified on the basis ofmass spectra as detailed in FIG. 6. The mass-to-charge (m/z) value (M)followed by retention time (T) in seconds is shown for each compound onthe horizontal axis. (G) Proposed pathway for noscapine biosynthesisbased on VIGS data. Solid arrows depict steps supported by VIGS data,dotted arrows depict additional proposed steps. For the secoberbineintermediates, R1=H or OH, R2=H or OH and R3=CH2OH or CHO or COOH (FIG.6). The noscapine structure is numbered according to the IUPACconvention;

FIG. 5: The ten genes exclusively expressed in the HN1 variety occur inthe genome of HN1 but are absent from that of varieties HT1 and HM1. (A)Amplification of fragments from the ten genes exclusively expressed inHN1 using two different primer pairs. (B) Amplification of fragments ofgenes from the protoberberine and morphinan branch pathways that areexpressed in all three varieties. Primers used are detailed in Table 3;HyperLadder I (Bioline Reagents, London, UK) was used as molecular sizestandard;

FIG. 6. Evidence for putative identities of intermediates from VIGSexperiments. All panels show the mass spectra of the pseudomolecularparent ion at the chromatographic peak apex in black and correspondingMS2 fragmentation spectra in red, scaled to relative abundance. MS2spectra were generated by targeting the parent ion with a isolationwidth of 3 m/z and using collisional isolation dissociation energy setto 35%. All mass spectra were obtained at a resolution setting of 7500.Text printed above selected diagnostic ions indicate the exactmonoisotopic mass of the ion, the calculated formula within limitsC=1:100, O=0:200, N=0:3 and H=1:200, and the number/total number offormulae returned within a 5 ppm error window. Fragments were reconciledagainst theoretical fragments generated by submitting candidate parentstructures to Mass Frontier software (version 5.01.2; HighChem,Bratislava, Slovakia). Candidate parent structures were derived fromPubChem searches and the comprehensive review of Papaver spp. alkaloids(Sariyar (2002) Pure Appl. Chem. 74, 557-574). (A)Tetrahydrocolumbamine; this compound was characterized from a peakeluting at 174s from VIGS-silenced CYP719A21. Eight out of ten observedMS2 fragments were calculated as feasible by Mass Frontier; only the twomost abundant diagnostic fragments are shown. (B) Secoberbineintermediate 1 (C21H25NO6); this compound was characterized from a peakeluting at 147s from VIGS-silenced CYP82X2. If R1=OH, R2=H, andR3=CH2OH, then this compound is narcotolinol which is consistent withboth annotated fragments. Another candidate formula fit would bedemethoxylated narcotindiol (R1=H, R2=OH, R3=CH2OH); however thisstructure would not form the observed fragment at 206.0816. (C)Secoberbine intermediate 2 (C21H23NO6); this compound was characterizedfrom a peak eluting at 103s from VIGS-silenced CYP82X2. If R1=OH, R2=H,and R3=CHO, then this compound would be a desmethylated derivative ofmacrantaldehyde. (D) Papaveroxine; this compound was characterized froma peak eluting at 214s from VIGS-silenced PSCXE1. The 398.1600 fragmentobserved is consistent with deacetylation. (E) Narcotinehemiacetal; thiscompound was characterized from a peak eluting at 121s fromVIGS-silenced PSSDR1. (F) Narcotoline (4′-desmethylnoscapine); thiscompound was characterized from a peak eluting at 208s fromVIGS-silenced PSMT2. Other isobaric possibilities were 6- or7-desmethylnoscapine. However, the 206.0816 fragment observed isconsistent with a hydroxylated 4′ position. Alternative structures couldbe discounted by comparing the candidate fragmentation spectra with thatfrom synthetic 7-desmethylnoscapine, which eluted at a differentretention time and lacked the characteristic 206.0816 fragment;

Table 1 Illustrates the % identity of CYP82Y1, PSCXE1, PSDFR1 and PSAT1(SEQ ID 17-20) with their respective closest functionally characterisedhomologues. Accession numbers given are from GenBank, Swiss-Prot or PDBdatabases;

Table 2. Genotyping of F3 families derived from two F2 phenotypicclasses: low noscapine and high noscapine. The observed versus expectedsegregation ratios strongly support the hypothesis that individuals inthe low noscapine F2 class are heterozygous for the HN1 gene cluster andindividuals in the high noscapine class are homozygous;

Table 3. Primer sequences and associated information.

TABLE 1 % Accession Protein Identity number Annotation CYP82Y1 54CYP82X1 from Papaver somniferum 48 CYP82X2 from Papaver somniferum 39ABM46919.1 CYP82E3, nicotine demethylase from Nicotiana tomentosiformisPSCXE1 45 2O7R_A AeCXE1, Carboxyl esterase from Actinidia erianthaPSSDR1 46 AAB41550.1 Vestitone reductase from Medicago sativa 45ABQ97018.1 Dihydroflavonol 4-reductase from Saussurea medusa PSAT1 66Q94FT4.1 Salutaridinol 7-O-acetyltransferase from Papaver somniferum

TABLE 2 Expected segregation in F3 if F2 low noscapine class is F3 seedObserved heterozygous family segregation and the high Noscapine class(obtained Number of gene noscapine and genotyping through self- of F3cluster in F3 class is result of F2 pollination of individuals progenyhomozygous Chi-Square individual F2 individual) genotyped GC+ GC− GC+GC− X-squared p-value low noscapine/GC+ S-111809 28 18 10 21 7 1.7140.190 low noscapine/GC+ S-111835 26 18 8 19.5 6.5 0.462 0.497 highnoscapine/GC− S-111714 28 28 28 high noscapine/GC− S-111854 54 54 54

TABLE 3 Primer sequences (5′- to 3′-) Gene Forward Reverse NotesApplication PSMT1 GATTCCCGATTTACTCC AACACAAAATACGATTACTT primer pair 1Primersfor the TGATG ACTTTTGTCC amplification PSMT1 TGCCTCATGTTATTTCTGCATGAAATGGATGTAGTTA primer pair 2 of fragments GTTGCC TCTTGGfrom genomic PSMT2 ATTGATGTCGGTGGTGG ATTCCCGTTCAAGTAAACAT primer pair 1DNA of HM1,HT1 TCACG GCGG and HN1 as shown PSMT2 GCAACTGTTTCATTAACCAGTAAATTCACACATTCCG primer pair 2 in FIG. 5 AGGCACATCC TATCTTCCC PSMT3GCTTCAGCATTGGTTAA GAGGGTAAGCCTCAATAACA primer pair 1 CGAGTGC GACTGGPSMT3 AGACCGTTTGTACCGAA TCGTTCCATTCGTGAAGAAT primer pair 2 TTCTGC GCCYP82X1 GAACCATTAAACACTTG TGCAATTGAATTTAGCTCAT primer pair 1 AGTCATGCCTCC CYP82X1 TTGATGAACGACAAGGA ATTCATGATTGTGACCTTTG primer pair 2 ACCGTAATCC CYP82X2 ATGTGGAAAACGGTAAG ACGATTCTGTCATCATCATT primer pair 1CAAGTGG TTCGC CYP82X2 CAACCTCAATCTAGCTA CCCAAGATTTTCATATCCTTprimer pair 2 GAGTCG TACAA CYP82Y1 CAATAATTGAGTAATTTGCTCCGTAAGTGCTCCTGTG primer pair 1 CAGTTCATTCATGG CYP82Y1GAATTGTGGTAAAAAAT CCCTTCACATCTACCATCC primer pair 2 TAGATGCAG CTTCYP719A21 CAAAGAGTCAATCTGAC CGAGTGCCCATGCAGTGG primer pair 1 TCAAGCTAGCCYP719A21 TCAAACCCTGCTACTAA CACTCCATCAGACACACAAG primer pair 2CACTTACTTGC ACC PSAT1 TTTTATCGACCTTGAGG AAATGGCAGTTCCACCGC primer pair 1AACAATTAGG PSAT1 GACTTCATGATGAAATC CACTGCTGACTTCCATATC primer pair 2AGATGCAC AAAGC PSCXE1 ATGCTGTTGATGCTTTA AGCTGAATTTGTCGATCAAprimer pair 1 AACTGGG TAAGTGG PSCXE1 AATAAAAATCCAACAATACTGGCATGATATGCAACA primer pair 2 GGCAGATCC TTAGC PSSDR1GGAAGATGTGAGCCACC GATACACTGGGAGGAGGAT primer pair 1 TTAAAGC GGG PSSDR1GAGAGTAACCACATCTT CGGCAAAATTCATTCCTTG primer pair 2 TGTTGTCGG AGC BBEGTTTACTCCCACGTGCA CATTCCTCGTCTAATTCATC TC TGC TNMT GTTTACTCCCACGTGCAGCTTCACTACTTCTTCTTG TC AAAAG SalR AAACAATGCTGGGGTTGCCATTATAATTTCCAATGCCGT AGTTC SalAT TAAGAGAGGGAGACCAC CATTCGTTGTTGTTGCTGGAG GTAAG T6ODM CTTATGAAGCTAGGTAA CATCCTCATTGCTTGTGT TGGTATGGA CC PSMT1CTCTAAAATGCCAAACG sequencing primer Primers used as CGsequencing primers PSMT1 GACCCTTTGGGACTTCC sequencing primerto obtain genomic TCG DNA sequence from PSMT1 CGTGTTGTTTGGTCCCTsequencing primer HN1 CG PSMT1 TGCCTCATGTTATTTCT sequencing primerGTTGCC PSMT1 GATTCCCGATTTACTCC sequencing primer TGATGG PSMT1AACACAAAATACGATTA sequencing primer CTTACTTTTGTCC PSMT1TGCCTCATGTTATTTCT sequencing primer GTTGCC  PSMT1 GCATGAAATGGATGTAGsequencing primer TTATCTTGG PSMT1 AAATCGTTCGCTCTTTA sequencing primerCCGC PSMT1 CACACCAAACTTGATCA sequencing primer TTGTC PSMT2ATTGTTGATATTGAATC sequencing primer AGAAACTTTC PSMT2 TCAATACCAGTACTGTTsequencing primer AGTTTCCG PSMT2 GCAACTGTTTCATTAAC sequencing primerAGGCACATCC PSMT2 ATTGATGTCGGTGGTGG sequencing primer TCACG PSMT2GCACACTGTCTTTTTCT sequencing primer TCCACC PSMT2 ACCGGAATGAGAATGCAsequencing primer TAAAGTAAAGG PSMT2 CCAATACCCAATCAATT sequencing primerAAACTC PSMT2 CAGTAAATTCACACATT sequencing primer CCGTATCTTCCC PSMT3ATTGTATAGCCAAAGTT sequencing primer GCAGGTAGGG PSMT3 AGACCGTTTGTACCGAAsequencing primer TTCTGC PSMT3 GCAGTGAAAGCCATATC sequencing primerCAAAGC PSMT3 AACCGTCCCCAAGATGA sequencing primer TTCC PSMT3TCGTTCCATTCGTGAAG sequencing primer AATGC PSMT3 GAGGGTAAGCCTCAATAsequencing primer ACAGACTGG CYP82X1 GAACCATTAAACACTTG sequencing primerAGTCATGC CYP82X1 TTGATGAACGACAAGGA sequencing primer ACCG CYP82X1TCGACAGCGGTTACGAA sequencing primer CG CYP82X1 CAATTATCAAAGAATCAsequencing primer ATGC CYP82X1 TGCAATTGAATTTAGCT sequencing primerCATCT  CYP82X1 ATTCATGATTGTGACCT sequencing primer TTGTAATCC CYP82X1GACAGAGGGCCCAAGTT sequencing primer AAGG CYP82X1 AGCAAACCATTCGTCCAsequencing primer TCC CYP82X1 TACGACAGGTTGCTAGC sequencing primer TTGGCYP82X2 AATAATGGATCAGTCAC sequencing primer GGCTTCC CYP82X2AATCCATCAGATTTTCA sequencing primer ACCAGAGAGG CYP82X2 TGTCAGCCAACCATTCGsequencing primer TCCATCCTAAC CYP82X2 GGCTTCCCGGAGATGACsequencing primer CCAGATTTTAT CYP82X2 TTGTTATTTTCATGACTsequencing primer ATTACCACCAGCTTCCT CTTA CYP82X2 AGTGGAGGAGGCACAAAsequencing primer AGTTAGGATGGAC CYP82X2 CCATGTCTGATAAATACsequencing primer GGGTCGGTGTTC CYP82X2 TTGTTGATAAGGACGACsequencing primer TAAGAATAAGCAGAAGA TA CYP82X2 ACGATTCTGTCATCATCsequencing primer ATTTTCGC CYP82X2 AGTCGTGTATCGTTCGC sequencing primerTTAATGC CYP82X2 CATGCCTATCTATTTCC sequencing primer TCCCTTGCCCTC CYP82X2TGTCAGCCAACCATTCG sequencing primer TCCATCCTAAC CYP82X2TGTTCGATCACGTTGTC sequencing primer TCTTTTTGCCATAA CYP82X2TAACAATAAAAGTACTG sequencing primer ATAATGGTGGTCGAAGG AGAA CYP82Y1TATTGATGTGGACCAGT sequencing primer ACC CYP82Y1 TGTAACTCTTGGTCACAsequencing primer TGG CYP82Y1 CGCGTACTTGACATTTA sequencing primer ACGCYP82Y1 GGATCATCGCCAAAAGA sequencing primer AAC CYP719A21CAAAGAGTCAATCTGAC sequencing primer TCAAGCTAGC CYP719A21TGAAATGCCTGAGATCA sequencing primer CTAAAATCG CYP719A21TCAAACCCTGCTACTAA sequencing primer CACTTACTTGC CYP719A21TGTAAAGACACTTCATT sequencing primer GATGGGC CYP719A21 TTCGATTTGTGTAAACAsequencing primer TTAATGATATTTGG CYP719A21 GAGATGATCAAGTGGTTsequencing primer TAACCATTCC CYP719A21 CGAGTGCCCATGCAGTGGsequencing primer PSCXE1 AATAAAAATCCAACAAT sequencing primer GGCAGATCCPSCXE1 ATGCTGTTGATGCTTTA sequencing primer AACTGGG PSCXE1GGTTAATCGAGAGATGT sequencing primer TTTGTGGTAGG PSCXE1 CGATGACACAGAGCAAGsequencing primer AACGAC PSCXE1 CGCGGGTATATGTGTAG sequencing primerCAATCG PSCXE1 CGGCAACGCCAGTTCCC sequencing primer PSSDR1CTAACAGGCAAACAATA sequencing primer ACAGGTTGC PSSDR1 GGAAGATGTGAGCCACCsequencing primer TTAAAGC PSSDR1 AAAGGTACTGACAGAAA sequencing primerGAGCTTGCC PSSDR1 AGATACACTGGGAGGAG sequencing primer GATGGG PSSDR1CGGCAAAATTCATTCCT sequencing primer TGAGC PSSDR1 AACATATAGCCAAAGGAsequencing primer CTCTTCG PSAT1 AGGATACACAATGACCC sequencing primer AACPSAT1 TTTTATCGACCTTGAGG sequencing primer AACAATTAGG PSAT1TGTTCACTAGGTGGAAA sequencing primer GAG PSAT1 AGTACAATACCGAGAAAsequencing primer TCCGACAAG PSAT1 GCTCAATTAATGGAACA sequencing primerGTAGTTACCC specific PCR conditions: PsMT1 VIC®-CGTGTTGTTTGGGCACACTGTCTTTTTCTTC 30 cylces, 20 s extension Primer pairs for TCCCTCGCACC at 72° genotyping of the PsMT2 VIC®-GCAACTGITTCAGCCAGCGCTAATACAAGGA 36 cylces, 50 s extension F2 mapping popula-TTAACAGGCACATCC TGTGG at 72° tion PsMT3 VIC®-GCAGTGAAAGCCTCGTTCCATTCGTGAAGAA 30 cylces, 30 s extension ATATCCAAAGC TGC at 72°CYP82X1 VIC®-GCTACGAAAGAT AGCAAACCATTCGTCCATCC 30 cylces, 30 s extensionAATGGTGCAGC at 72° CYP82X2 VIC®-ATGTGGAAAACG ACGATTCTGTCATCATCAT30 cylces, 50 s extension GTAAGCAAGTGG TTTCGC at 72° CYP719A21VIC®-TGAAATGCCTGA GGAATGGTTAAACCACTTG 30 cylces, 30 s extensionGATCACTAAAATCG ATCATCTC at 72° PSCXE1 VIC®-ATGCCAGTTTAAGGGAACTGGCGTTGCCG 30 cylces, 30 s extension GAGCAATAGAAATGG at 72°PSSDR1 VIC®-GAAGATGTGAGC GCTCAAGGAATGAATTTTG 30 cylces, 30 s extensionCACCTTAAAGC CCG at 72° CYP82X2 GTTGACGCAGGAAGCTT GGAACATAAGATTTAACTCPrimer pair for PCR amplification TTGC CGCCTCof the BAC library screening probe PSMT1 aaactcgagaagctTGGaaaggtaccCATGTACTAC Primer pairs for TCATAATCATCAATCAG TACATCATCTCCthe amplification PSMT2 aaactcgagaagcttGT aaaggtaccACTTGAATATand cloning of GTAACTAAGCCAGCGC ATCACCGC fragments selected CYP82X1aaaggatccTTTGAGTA aaaggtaccAACATCTACT for VIGS ATGGTGAAAAGA CTCGAGGATTGCYP82X2 aaactcgagaagcttTA aaaggtaccTTAACTCCGC GGAGGGTATGTCCGGC CTCGGCTCCCYP82Y1 aaaggatccTTCAGTT aaaggtaccGTTCATAGTAA CATTCATGGCG ATAATAACAGGCGCYP719A21 aaactcgagaagcttAT aaaggtaccCCAACAGGCCA GATCATGAGTAACTTATTTCCGTTG GGA PSCXE1 aaaggatccTGGCAGAT aaaggtaccTTATGATAGGA CCTTATGAATTCCAGCAGCTTATTC PSSDR1 aaaggatccGAAATTGAC aaaggtaccCATTCAAAAAC GAGACAATATGGGAATATGTGTGC PSAT1 aaaggatccCCTAAGAGA aaaggtaccAATACAAGTAT GATCCTCCAACTGGAAAACAAGAGAATAA PSPDS GAGGTGTTCATTGCCATG GTTTCGCAAGCTCCTGCATA TCAA GT

Materials and Methods

Plant Material

Three GSK Australia poppy varieties that predominantly accumulate eithernoscapine (High Noscapine, HN1), morphine (High Morphine, HM1) orthebaine (High Thebaine HT1), were grown in Maxi (Fleet) Rootrainers™(Haxnicks, Mere, UK) under glass in 16 hour days at the University ofYork horticulture facilities. The growth substrate consisted of 4 partsJohn Innes No. 2, 1 part Perlite and 2 parts Vermiculite. The HM1×HN1 F2mapping population was grown at the GlaxoSmithKline Australiafield-trial site, Latrobe, Tasmania from September 2009 to February2010.

Crossing and Selfing

Crosses were carried out between HN1 and HM1 individuals to generate F1hybrid seed. At the hook stage of inflorescence development, immaturestamens were removed from selected HN1 flower buds. HN1 stigmas werefertilized with pollen from synchronously developing HM1 flowers shortlyafter onset of anthesis. To prevent contaminating pollen from reachingthe receptive stigmas, emasculated flowers were covered with a muslinbag for four days after pollination. Both the F1 and F2 generations wereself-pollinated to produce F2 and F3 seed, respectively.Self-pollination was ensured by covering the flowers shortly beforeonset of anthesis with a muslin bag.

RNA Isolation and cDNA Synthesis

Upper stems (defined as the 2 cm section immediately underneath thecapsule) and whole capsules were harvested at two developmental stagesrepresented by 1-3 days and 4-6 days, after petal fall. Five plants wereused per developmental stage and cultivar. The material was ground to afine powder in liquid nitrogen using a mortar and pestle. RNA wasisolated from the powder using a CTAB-based extraction method (Chang etal (1993) Plant Mol. Biol. Rep. 11, 113-116) with small modifications:(i) three sequential extractions with chloroform:isoamylalcohol (24:1)were performed and (ii) the RNA was precipitated overnight with lithiumchloride at 4° C. After spectrophotometric quantification, equal amountsof RNA were pooled from five plants per cultivar, development stage andorgan. The pooled samples underwent a final purification step using anRNeasy Plus MicroKit (Qiagen, Crawley, UK). RNA was typically eluted in30-100 μl water. cDNA was prepared with the SMART cDNA LibraryConstruction Kit (Clontech, Saint-Germainen-Laye, France) according tothe manufacturer's instructions but using SuperScript II ReverseTranscriptase (Invitrogen, Paisley, UK) for first strand synthesis. TheCDSIII/3′PCR primer was modified to: 5′ ATT CTA GAT CCR ACA TGT TTT TTTTTT TTT TTT TTT TVN 3′ where R=A or G, V=A, C or G; N=A/T or C/G.Following digestion with MmeI (New England Biolabs, Hitchin, UK) thecDNA was finally purified using a QIAquick PCR Purification kit (Qiagen,Crawley, UK).

cDNA Pyrosequencing: Pyrosequencing was performed on the Roche 454GS-FLX sequencing platform (Branford, Conn.) using cDNA prepared fromthe following four samples of each of the three varieties:

-   -   i. upper stem, 1-3 days after petal fall    -   ii. upper stem, 4-6 days after petal fall    -   iii. capsule, 1-3 days after petal fall    -   iv. capsule, 4-6 days after petal fall

Raw Sequence Analysis, Contiguous Sequence Assembly and Annotation

The raw sequence datasets were derived from parallel tagged sequencingon the 454 sequencing platform (Meyer et al (2008) Nature Prot. 3,267-78). Primer and tag sequences were first removed from all individualsequence reads. Contiguous sequence assembly was only performed onsequences longer than 40 nucleotides and containing less than 3% unknown(N) residues. Those high quality Expressed Sequence Tag (EST) sequenceswere assembled into unique contiguous sequences with the CAPS SequenceAssembly Program (Huang and Madan (1999) Genome Res. 9, 868-877), andthe resulting contigs were annotated locally using the BLAST2 program(Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402) against thenon-redundant peptide database downloaded from the NCBI.

Expression profiling: The number of ESTs associated with a specificconsensus sequence representing each of the candidate genes detailed inFIG. 1 was counted for each EST library. EST numbers were normalised onthe basis of total number of ESTs obtained per library. For eachvariety, EST counts were combined for the two developmental stages fromboth stems and capsules. Differences in candidate gene expression levelsbetween organs and varieties were visualised as a heat map usingMicrosoft Excel.

Preparation of Genomic DNA from Glasshouse Grown Plants

In order to amplify and obtain genomic sequences of the candidate genes30-50 mgs of leaf material was collected from 4-6 week oldglasshouse-grown seedlings from each of the three varieties. Genomic DNAwas extracted using the BioSprint 96 Plant kit on the BioSprint 96Workstation (Qiagen, Crawley, UK) according to the manufacturer'sprotocol. Extracted DNA was quantified using Hoescht 33258 andnormalized to 10 ng/ul.

Amplification and Sequencing of Candidate Genes from Genomic DNA

Primers for amplification and Sanger-sequencing of the candidate genesfrom genomic DNA were based on the respective contiguous sequencesassembled from the ESTs or on BAC sequences. The primer sequences areshown in Table 3. PCR amplifications were performed on pools of genomicDNA comprising DNA from four individuals. Amplification was typicallycarried out on 10 ng genomic DNA in 1× Phusion High Fidelity Buffersupplemented with 200 nM forward and reverse primers, 0.2 mM dNTPs, 0.02units/μl Phusion Hot Start DNA Polymerase (Finnzymes, Vantaa, Finnland).Standard PCR conditions were used throughout with annealing temperaturesand times dependent on primers and PCR equipment.

DNA Extraction from the Field-Grown F2 Mapping Population

40-50 mg of leaf tissue was harvested from F2 plants at the ‘smallrosette’ growth stage (˜10 leaves present on each plant) into 1.2 mlsample tubes. A 3 mm tungsten carbide bead was added to each tube andsamples were kept at −80° C. for a minimum of two hours prior tofreeze-drying for 18 hours. Following freeze drying, samples werepowdered by bead-milling (Model TissueLyser, Qiagen, Hilden, Germany) at30 Hz for two 60 s cycles separated by plate inversion. DNA extractionwas performed with the Nucleospin Plant II kit (Macherey-Nagel, Duren,Germany) using the supplied Buffer Set PL2/3 following themanufacturer's protocol for centrifugal extraction. DNA was quantifiedby UV-spectroscopy.

Genotyping of the HN1×HM1 F2 Mapping Population for the Presence orAbsence of the HN1-Specific Candidate Genes

Plants of the F2 mapping population were genotyped for the presence orabsence of eight candidate genes. The gene primer pairs (Table 3) weredesigned with fluorescent tags (5′-VIC®-labeled) for use on the ABI3730xl capillary apparatus (Applied Biosystems, Foster City, Calif.).PCR amplifications were typically carried out on 10 ng genomic DNA in1×GoTaq buffer supplemented with 1 mM MgCl₂, 500 nM forward and reverseprimer, 0.125 mM dNTPs, 0.1 U GoTaq (Promega, Southampton, UK). Theamplification conditions were: 1 min 94° C., 30-36 cycles of 30 sdenaturation at 94° C., 30 s annealing at 62° C. and 20-50 s extensionat 72° C., followed by a final extension for 5 min at 72° C. Cyclenumber and extension times depended on the candidate gene (Table 3).Amplification products were diluted 1:20 in H₂O and fractionated on anABI 3730xl capillary sequencer (Applied Biosystems, Foster City,Calif.). Data were scored using GeneMarker™ software (Softgenetics,State College, Pa.).

Poppy Straw Analysis from Field Grown F2 Plants

Poppy capsules were harvested by hand from the mapping population oncecapsules had dried to approximately 10% moisture on the plant. Aftermanually separating the seed from the capsule, the capsule straw samples(Poppy Straw) were then ground in a ball mill (Model MM04, Retsch, Haan,Germany) into a fine powder. Samples of ground poppy straw were thenweighed accurately to 2±0.003 g and extracted in 50 ml of a 10% aceticacid solution. The extraction suspension was shaken on an orbital shakerat 200 rpm for a minimum of 10 min, then filtered to provide a clearfiltrate. The final filtrate was passed through a 0.22 μm filter priorto analysis. The loss on drying (LOD) of the straw was determined bydrying in an oven at 105° C. for 3 hours.

All solutions were analysed using a Waters Acquity UPLC system (WatersLtd., Elstree, UK). fitted with a Waters Acquity BEH C18 column, 2.1mm×100 mm with 1.7 micron packing. The mobile phase used a gradientprofile with eluent A consisting of 10 mM ammonium bicarbonate of pH10.2 and eluent B methanol. The mobile phase gradient conditions usedare as listed in the table below with a linear gradient. The flow ratewas 0.5 ml per minute and the column maintained at 60° C. The injectionvolume was 2 μl and eluted peaks were ionised in positive APCI mode anddetected within 5 ppm mass accuracy using a Thermo LTQ-Orbitrap. Theruns were controlled by Thermo Xcalibur software (Thermo FisherScientific Inc., Hemel Hempstead, UK).

Gradient Flow Program:

TIME (minutes) % Eluent A % Eluent B Flow (ml/min) 0.0 98. 2.0 0.50 0.298.0 2.0 0.50 0.5 60.0 40 0.50 4.0 20.0 80.0 0.50 4.5 20.0 80.0 0.50

Mass spectra were collected over the 150-900 m/z range at a resolutionsetting of 7500. All data analysis was carried out in the R programminglanguage in a 64-bit Linux environment (R 2.11). Peak-picking wasperformed using the Bioconductor package, XCMS (Smith et al (2006) Anal.Chem. 78, 779-787), employing the centWave algorithm (Tautenhahn et al(2008) BMC Bioinformatics 9, 504). Redundancy in peak lists was reducedusing the CAMERA package (Kuhl et al (2012) Anal. Chem. 84, 283-289).Alkaloids were identified by comparing exact mass and retention timevalues to those of standards and quantified by their pseudomolecular ionareas using custom R scripts.

Bacterial Artificial Chromosome (BAC) Library Construction

The HN1 BAC library was constructed from high molecular weight (HMW)genomic DNA processed at Amplicon Express, Inc. (Pullman, Wash.) fromfour week old seedlings using the method described (Tao et al (2002)Theor. Appl. Genet. 105, 1058-1066). The HMW DNA was partially digestedwith the restriction enzyme HindIII and size selected prior to ligationof fragments into the pCC1BAC vector (Epicentre Biotechnologies,Madison, Wis.) and transformation of DH10B E. coli cells, which werethen plated on Luria-Bertani (LB) agar with chloramphenicol, X-gal andIPTG at appropriate concentrations. Clones were robotically picked witha Genetix QPIX (Molecular Devices, Sunnyvale, Calif.) into 240 384-wellplates containing LB freezing media. Plates were incubated for 16 hours,replicated and then frozen at −80° C. The replicated copy was used as asource plate for nylon filters that were made and used for screeningusing the PCR DIG Probe Synthesis Kit (Roche Applied Science,Indianapolis, Ind.). To estimate insert sizes, DNA aliquots of 10 BACminipreps were digested with 5 U of NotI enzyme for 3 hours at 37° C.The digestion products were separated by pulsed-field gelelectrophoresis (CHEF-DRIII system, Bio-Rad, Hercules, Calif.) in a 1%agarose gel in TBE. Insert sizes were compared to those of the LambdaLadder MidRange I PFG Marker (New England Biolabs, Ipswich, Mass.).Electrophoresis was carried out for 18 hours at 14° C. with an initialswitch time of 5 s, a final switch time of 15 s, in a voltage gradientof 6 V/cm. The average BAC clone size for the library was found to be150 Kb.

Filter Construction and Screening

Filter design and screening was carried out at Amplicon Express, Inc.(Pullman, Wash.). Bioassay dishes containing LB agar plate media and12.5 μg/mL chloramphenicol were prepared. Positively charged nylonAmersham Hybond-N⁺ membrane (GE Healthcare Bio-Sciences, Piscataway,N.J.) was applied to the media surface and the GeneMachines G3 (GenomicsSolutions, Bath, UK) was used to robotically grid 18,432 clones induplicate on filters. The filters were incubated at 37° C. for 12 to 14hours. The filters were processed using the nylon filter lysis method(Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001, ed. 3, vol. 1,chap. 1) with slight modifications. Following processing, the DNA waslinked to the hybridization membrane filters according to the Hybond N+manual by baking at 80° C. for 2 hours. To screen the library a 643 bpdigoxigenin (DIG)-labeled probe representing position 2161-2803 in thegenomic sequence of CYP82X2 (SEQ ID NO 6) was generated from 1.5 ng gDNAby PCR reaction using the primers shown in Table 3 and the PCR DIGsynthesis kit (Roche Applied Science, Indianapolis, Ind.) according tothe manufacturer's instructions. A non-labeled probe was amplified,diluted and spotted to each filter in the following dilutions of 2 ng, 1ng, 0.1 ng and 0.0 ng as a positive control. The controls were baked at80° C. for 30 min. Following a 30 min prehybridizing wash in DIG EasyHybsolution at 45° C. approximately 0.5 μl of denatured DIG labeled PCRproduct was added per ml of hybridization solution with the nylonfilters and incubated with gentle shaking overnight at 45° C. The nylonfilters were washed twice in a 2× standard sodium citrate (SSC), 0.1%sodium dodecyl sulfate (SDS) buffer at room temperature for 5 min each,and twice with a 0.5×SSC, 0.1% SDS buffer at 65° C. for 15 minutes each.The hybridized probe was detected using NBT/BCIP stock solutionaccording to the manufacturer's instructions (Roche Applied Science,Indianapolis, Ind.) and was found to hybridize to six BAC clones.

BAC sequencing and automated sequence assembly: The six positive BACclones from the BAC library were sequenced at Amplicon Express, Inc.(Pullman, Wash.) by Focused Genome Sequencing (FGS) with an averagedepth of 100× coverage. FGS is a Next Generation Sequencing (NGS) methoddeveloped at Amplicon Express that allows very high quality assembly ofBAC clone sequence data using the Illumina HiSeq platform (Illumina,Inc, San Diego, Calif.). The proprietary FGS process makes NGS taggedlibraries of BAC clones and generates a consensus sequence of the BACclones with all reads assembled at 80 bp overlap and 98% identity. Thegapped contiguous sequences were ordered and orientated manually basedon mate pair sequences from four libraries of insert size 5000, 2000,500 and 170 bp. Overlapping BAC clones, PS_BAC193L09, PS_BAC179L19,PS_BAC150A23 and PS_BAC164F07, which together encoded all 10 genes fromthe HN1 cluster, were selected for further sequence assembly. Wherepossible, gaps and ambiguous regions on both BAC clones were covered byprimer walking with traditional Sanger sequencing to validate theassembly. Combination of the four overlapping BAC sequences gave asingle continuous consensus sequence assembly of 401 Kb. The sequencesof the 10 genes from the HN1 cluster were determined independently bySanger sequencing and the 100% agreement of the Sanger determined genesequences with the assembly from FGS provided quality assurance for thewhole assembly.

Annotation of the assembled sequence: The sequences of the four BACclones were annotated with an automated gene prediction program FGENESH(Salamov and Solovyev (2002) Genome Res. 10, 516-522). The genestructure including exon-intron arrangement for the 10 genes in the HN1cluster was validated by comparison with cDNA sequence for each gene.cDNA sequence was not available for any of the remaining ORFs detailedin FIG. 3 since they are not represented in any of the EST libraries.The predicted function of all ORFs was evaluated by BLAST analysis(Altschul et al (1997) Nucleic Acids Res. 25, 3389-3402) and those ORFswith significant hits (e-value less than 1e⁻⁸) were included in FIG. 3.

Generation of Plasmid Constructs for Virus Induced Gene Silencing (VIGS)

The tobacco rattle virus (TRV) based gene silencing system (Liu et al(2002) Plant J. 30, 415-422) was used to investigate the gene functionof PSMT1, PSMT2, CYP719A21, CYP82X2, PSSDR1 and PSCXE1. DNA fragmentsselected for silencing were amplified by PCR and cloned into thesilencing vector pTRV2 (GenBank accession no: AF406991). They werelinked to a 129 bp-long fragment (SEQ ID NO: 30) of the P. somniferumPHYTOENE DESATURASE gene (PSPDS) in order to simultaneously silence therespective candidate genes and PSPDS. Plants displaying thephoto-bleaching phenotype resulting from PSPDS silencing (Hileman et al(2005) Plant J. 44, 334-341) were identified as plants successfullyinfected with the respective silencing constructs and selected forfurther analysis.

Generation of the pTRV2:PDS construct: A 622 bp fragment of PSPDS wasamplified from cDNA prepared from HN1 using primers shown in Table 3.Sau3Al digestion of the 622 bp PCR product yielded among others afragment of 129 bp (SEQ ID NO: 30) which was cloned into the BamHI siteof the pTRV2 vector. The orientation and fidelity was confirmed bysequencing and the resulting pTRV2:PDS vector was used in the generationof the VIGS construct for each candidate gene. The pTRV2:PDS constructalso served as the control in the VIGS experiments.

DNA fragments selected for silencing the respective candidate genes wereamplified from either HN1 genomic or cDNA. Primers used foramplification as well as the positions of the selected sequences withinthe respective open reading frames are shown in Table 3. The PSMT1,CYP719A21 and CYP82X2 fragments were first cloned into pTV00 (Ratcliffet al (2001) Plant J., 237-245) using HindIII and KpnI and thensubcloned into pTRV2:PDS using BamHI and KpnI. PSMT2, PSCXE1 and PSSDR1fragments were cloned directly into pTRV2:PDS using BamHI and KpnI. Theorientation and fidelity of all constructs was confirmed by sequencing.

Transformation of Agrobacterium tumefaciens with VIGS constructs: VIGSconstructs were propagated in E. coli strain DH5α and transformed intoelectrocompetent Agrobacterium tumefaciens (strain GV3101) byelectroporation.

Infiltration of plants: Separate overnight liquid cultures of A.tumefaciens containing individual VIGS constructs (each consisting of aselected DNA fragment from the target gene linked to the 129 bp-longfragment from the P. somniferum PHYTOENE DESATURASE gene) were used toinoculate LB medium containing 10 mM MES, 20 μM acetosyringone and 50μg/ml kanamycin. Cultures were maintained at 28° C. for 24 hours,harvested by centrifugation at 3000×g for 20 min, and resuspended ininfiltration solution (10 mM MES, 200 μM acetosyringone, 10 mM MgCl₂) toan OD₆₀₀ of 2.5. A. tumefaciens harbouring the individual VIGSconstructs including the control, pTRV2:PDS, were each mixed 1:1 (v/v)with A. tumefaciens containing pTRV1 (GenBank accession no: AF406990),and incubated for two hours at 22° C. prior to infiltration. Two weekold seedlings of HN1 grown under standard greenhouse conditions (22° C.,16 h photoperiod), with emerging first leaves, were infiltrated asdescribed (Hagel and Facchini (2010) Nat. Chem. Biol. 6, 273-275).

Latex and capsule analysis of silenced plants: Leaf latex of infiltratedplants displaying photo-bleaching as a visual marker for successfulinfection and silencing was analyzed when the first flower buds emerged(˜7 week old plants). Latex was collected from cut petioles, with asingle drop dispersed into 500 μl of 10% acetic acid. This was diluted10× in 1% acetic acid to give an alkaloid solution in 2% acetic acid forfurther analysis. Capsules were harvested from the same plants used forlatex analysis and single capsules were ground to a fine powder in aball mill (Model MM04, Retsch, Haan, Germany). Samples of ground poppystraw were then weighed accurately to 10±0.1 mg and extracted in 0.5 mlof a 10% acetic acid solution with gentle shaking for 1 h at roomtemperature. Samples were then clarified by centrifugation and a 50 μlsubsample diluted 10× in 1% acetic acid to give an alkaloid solution in2% acetic acid for further analysis. All solutions were analyzed asdescribed for the poppy straw analysis from field grown F2 plants.Likewise, all data analysis was carried out using the R programminglanguage. Putative alkaloid peaks were quantified by theirpseudomolecular ion areas using custom scripts. Peak lists were compiledand any peak-wise significant differences between samples wereidentified using 1-way ANOVA with p-values adjusted using the Bonferronicorrection for the number of unique peaks in the data set. For anypeak-wise comparisons with adjusted p-values <0.05, Tukey's HSD test wasused to identify peaks that were significantly different between anygiven sample and the control. Alkaloids were identified by comparingexact mass and retention time values to those of standards. Wherestandards were not available, the Bioconductor rcdk package (Smith et al(2006) Anal. Chem. 78, 779-787) was used to generate pseudomolecularformulae from exact masses within elemental constraints C=1 100, H=1200, O=0 200, N=0 3 and mass accuracy <5 ppm. The hit with the lowestppm error within these constraints was used to assign a putativeformula.

EXAMPLE 1 Transcriptomic Analysis Reveals the Exclusive Expression of 10Genes Encoding Five Distinct Enzyme Classes in a High NoscapineProducing Poppy Variety, HN1. These Genes are Absent from the Genome ofTwo Noscapine Non-Producing Varieties

Capsule extract from three opium poppy varieties developed in Tasmaniafor alkaloid production designated as High Morphine 1 (HM1), HighThebaine 1 (HT1) and High Noscapine 1 (HN1) on the basis of the mostabundant alkaloid in each case (FIG. 1A) underwent metabolite profiling.Noscapine was found to be unique to HN1 relative to HM1 and HT1. Roche454 pyrosequencing was performed on cDNA libraries derived from stem andcapsule tissue from all three varieties. Analysis of Expressed SequenceTag (EST) abundance led to the discovery of a number of previouslyuncharacterized genes that are expressed in the HN1 variety but arecompletely absent from the HM1 and HT1 EST libraries (FIG. 1B). Thecorresponding genes were putatively identified as threeO-methyltransferases (PSMT1, PSMT2, PSMT3), four cytochrome P450s(CYP82X1, CYP82X2, CYP82X3 and CYP719A21), an acetyltransferase (PSAT1),a carboxylesterase (PSCXE1) and a short-chain dehydrogenase/reductase(PSSDR1). In contrast a number of other functionally characterized genesassociated with benzylisoquinoline alkaloid synthesis, includingBerberine Bridge Enzyme (BBE), Tetrahydroprotoberberinecis-N-MethylTransferase (TNMT), Salutaridine Reductase (SalR),Salutaridinol 7-O-AcetylTransferase (SalAT) and Thebaine 6-O-demethylase(T6ODM) were expressed in all three varieties (FIG. 1B). PCR analysis ongenomic DNA from all three varieties revealed that the genes exclusivelyexpressed in the HN1 variety are present as expected in the genome ofHN1 but absent from the genomes of the HM1 and HT1 varieties (FIG. 1Band FIG. 5).

EXAMPLE 2 Analysis of an F2 Mapping Population Shows the Genes areTightly Linked in HN1 and their Presence is Associated with theProduction of Noscapine

An F2 mapping population of 271 individuals was generated using HN1 andHM1 as parents. Genotyping of the field grown F2 population revealedthat the HN1 specific genes are tightly linked and associated with thepresence of noscapine suggesting they occur as a gene cluster involvedin noscapine biosynthesis (FIG. 2B). Analysis of noscapine levels infield grown F2 capsules revealed that individuals containing thisputative gene cluster fall into two classes. The first class containing150 individuals, have relatively low levels of noscapine and the secondclass containing 63 individuals exhibit the high noscapine trait of theparental HN1 variety (FIG. 2B). The 58 F2 individuals that lack theputative gene cluster contain undetectable levels of noscapine (FIG.2B). F3 family analysis confirmed that F2 individuals exhibiting thehigh noscapine trait were homozygous for the gene cluster while thoseexhibiting the low noscapine trait were heterozygous (Table 2).Noscapine levels in both the F1 population (FIG. 2A) and theheterozygous F2 class are much lower than the intermediate levelsexpected for a semi-dominant trait, suggesting involvement of some formof repression. The step change to high noscapine in homozygous F2 classsuggests this trait is linked to the gene cluster locus rather thanspread quantitatively among other loci.

EXAMPLE 3 Bacterial Artificial Chromosome Sequencing Confirms that the10 Genes Exist as a Complex Gene Cluster

To Further Characterize the Putative Noscapine Gene Cluster, a BacterialArtificial Chromosome (BAC) Library was Prepared from Genomic DNAIsolated from HN1 and Six Overlapping BACs Containing Genes from theCluster were Identified.

Next generation and Sanger sequencing was used to generate a highquality assembly of 401 Kb confirming the arrangement of the 10 genes ina cluster spanning 221 Kb (FIG. 3). Only one other homologous gene, acarboxylesterase (PSCXE2), was found in the genomic sequence flankingthe gene cluster (FIG. 3) but PSCXE2 was not represented in any of ourEST libraries. Interspersed among the ten genes are both retrotransposonand DNA transposable element (TE) sequences (FIG. 3), which may havesome function in gene rearrangement for cluster formation as thought tobe the case for the thalianol and marneral clusters from A. thaliana(Field et al (2011) PNAS 108, 16116-16121).

EXAMPLE 4 Virus Induced Gene Silencing Results in Accumulation ofPathway Intermediates Allowing Gene Function to be Linked to NoscapineSynthesis and a Novel Bifurcated Biosynthetic Pathway to be Proposed

In order to functionally characterize the genes in the HN1 cluster VirusInduced Gene Silencing (VIGS) was performed on poppy seedlings. VIGS inpoppy seedlings persists through to mature plant stages (Hileman et al(2005) Plant J. 44, 334-341), and therefore both leaf latex and capsuleextracts were routinely assayed (FIG. 4). Silencing PSMT1 resulted inaccumulation of scoulerine in capsules and also low levels of reticulinein latex, indicating that this gene product is responsible for the firstcommitted step in the pathway to noscapine synthesis (FIG. 4A). Thepredicted product of PSMT1 is tetrahydrocolumbamine (FIG. 6), whichaccumulated in seedlings and capsules that were silenced for CYP719A21(FIG. 4B). CYP719A21 shows high homology to cytochrome P450 oxidasesthat act as methylenedioxy bridge-forming enzymes (Diaz Chavez et al(2011) Arch. Biochem. Biophys. 507, 186193; Ikezawa et al (2009) PlantCell Rep. 28, 123-133). Therefore CYP719A21 may encode a canadinesynthase. FIG. 6). Silencing of a second cytochrome P450 gene, CYP82X2,resulted in accumulation of several secoberbine intermediates some ofwhich may represent side products to the main synthetic pathway (FIG.4C, FIG. 6). Silencing of the carboxylesterase gene PSCXE1 resulted inaccumulation of up to 20% total alkaloid content of putativepapaveroxine (FIG. 6) implying acetylation of a secoberbine intermediateas depicted in FIG. 4G. The PSAT1 gene from the HN1 cluster is anobvious candidate for this reaction. Silencing of PSSDR1 resulted inaccumulation of what was putatively identified as narcotinehemiacetal(FIG. 6), an immediate precursor of noscapine (FIG. 4G). These datasupport a biosynthetic route to noscapine that involves earlyO-methylation of a secoberbine intermediate at the position equivalentto the C4′ hydroxyl group of noscapine (FIG. 4G). However, silencingPSMT2, resulted in accumulation of up to 20% narcotoline, indicatingthat O-methylation at the C4′ hydroxyl group can also occur as a finalstep in noscapine production (FIG. 4F). These results imply bifurcationof the main pathway at the secoberbine intermediate stage with PSMT2being responsible for both the O-methylation of a secoberbineintermediate and narcotoline. Silencing PSMT2 results in accumulation ofhigh levels of narcotoline as flux is directed down the desmethyl branchof the pathway (FIG. 4F).

1. An isolated nucleic acid molecule that comprises a gene cluster thatencodes two or more polypeptides involved in the biosynthesis of opiatealkaloids or intermediates, wherein one of said two polypeptides isencoded by a nucleotide sequence selected from the group consisting of:i) a nucleotide sequence as set forth in SEQ ID NO: 8; ii) a nucleotidesequence, wherein said sequence is degenerate as a result of the geneticcode to the nucleotide sequence defined in (i); iii) a nucleic acidmolecule, the complementary strand of which hybridizes under stringenthybridization conditions to the nucleotide sequence in SEQ ID NO: 8 andwhich encodes a polypeptide that has carboxylesterase activity; and iv)a nucleotide sequence that encodes a polypeptide comprising an aminoacid sequence as set forth in SEQ ID NO: 18 or a nucleotide sequencethat encodes a polypeptide that has 46% amino acid sequence identityacross the full length amino acid sequence set forth in SEQ ID NO: 18wherein said polypeptide has carboxylesterase activity.
 2. An isolatednucleic acid molecule that comprises a gene cluster that encodes two ormore polypeptides involved in the biosynthesis of opiate alkaloids orintermediates, wherein one of said two polypeptides is encoded by anucleotide sequence selected from the group consisting of; i) anucleotide sequence as set forth in SEQ ID NO: 9; ii) a nucleotidesequence, wherein said sequence is degenerate as a result of the geneticcode to the nucleotide sequence defined in (i); iii) a nucleic acidmolecule, the complementary strand of which hybridizes under stringenthybridization conditions to the sequence in SEQ ID NO: 9 and whichencodes a polypeptide that has short-chain dehydrogenase/reductaseactivity; and iv) a nucleotide sequence that encodes a polypeptidecomprising an amino acid sequence as set forth in SEQ ID NO: 19 or anucleotide sequence that encodes a polypeptide that has is 46% aminoacid sequence identity across the full length amino acid sequence setforth in SEQ ID NO: 19 wherein said polypeptide has short-chaindehydrogenase/reductase activity.
 3. The isolated nucleic acid moleculeaccording to claim 1, wherein said nucleic acid molecule furtherincludes a nucleotide sequence selected from the group consisting of i)a nucleotide sequence as set forth in SEQ ID NO: 9; ii) a nucleotidesequence, wherein said sequence is degenerate as a result of the geneticcode to the nucleotide sequence defined in (i); iii) a nucleic acidmolecule, the complementary strand of which hybridizes under stringenthybridization conditions to the sequence in SEQ ID NO: 9 and whichencodes a polypeptide that has short-chain dehydrogenase/reductaseactivity; and iv) a nucleotide sequence that encodes a polypeptidecomprising an amino acid sequence as set forth in SEQ ID NO: 19 or anucleotide sequence that encodes a polypeptide that has is 46% aminoacid sequence identity across the full length amino acid sequence setforth in SEQ ID NO: 19 wherein said polypeptide has short-chaindehydrogenase/reductase activity.
 4. The isolated nucleic acid moleculeaccording to claim 1, wherein the nucleic acid molecule further includesone or more nucleotide sequences selected from the group consisting of:i) a nucleotide sequence as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or10; ii) a nucleotide sequence, wherein said sequence is degenerate as aresult of the genetic code to the nucleotide sequence defined in (i);iii) a nucleic acid molecule, the complementary strand of whichhybridizes under stringent hybridization conditions to the sequenceshown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, wherein saidnucleic acid molecule encodes polypeptides involved in the biosynthesisof P. somniferum opiate alkaloids or intermediates in the biosynthesisof opiate alkaloids; iv) a nucleotide sequence that encodes apolypeptide comprising an amino acid sequence shown in SEQ ID NO: 11,12, 13, 14, 15, 16, 17 or 20; and v) a nucleotide sequence that encodesa polypeptide comprising an amino acid sequence, wherein said amino acidsequence is modified by addition, deletion or substitution of at leastone amino acid residue as represented in iv) above and which hasretained or enhanced opiate alkaloid biosynthetic activity.
 5. Thenucleic acid molecule according to claim 4, wherein said nucleic acidmolecule comprises a nucleotide sequence as shown in SEQ ID NO: 1, 2, or3, and wherein said nucleic acid molecule encodes a polypeptide withmethyl transferase activity. 6-7. (canceled)
 8. The nucleic acidmolecule according to claim 4, wherein said nucleic acid moleculecomprises a nucleotide sequence as shown in SEQ ID NO: 5, 6, 7, or 10,wherein said nucleic acid molecule encodes a polypeptide with cytochromeP450 activity. 9-12. (canceled)
 13. The nucleic acid molecule accordingto claim 1, wherein said nucleic acid molecule comprises SEQ ID NO: 8and 9 and further comprises one or more nucleotide sequences selectedfrom the group consisting of: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 and
 10. 14.The nucleic acid molecule according to claim 1, wherein said nucleicacid molecule comprises SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10 and furthercomprises a nucleotide sequence selected from the group consisting of:SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 and
 10. 15. The nucleic acidmolecule according to claim 1, wherein said nucleic acid moleculeincludes each of the nucleotide sequences as shown in SEQ ID NO: 1, 2,3, 4, 5, 6, 7, 8, 9 and
 10. 16. A vector comprising a nucleic acidmolecule according to claim
 1. 17. The vector according to claim 16wherein said vector is a bacterial artificial chromosome.
 18. Atransgenic cell transformed or transfected with the nucleic acidmolecule of claim
 1. 19. The cell according to claim 18 wherein saidcell is a plant cell.
 20. The cell according to claim 19 wherein saidplant cell is from the genus Papaver.
 21. The cell according to claim 20wherein said plant cell is a Papaver somniferum cell.
 22. A plantcomprising the plant cell of claim
 19. 23. The cell according to claim18, wherein said cell is a microbial cell.
 24. A nucleic acid moleculecomprising a transcription cassette, wherein said cassette includes oneor more nucleotide sequences shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,9 and 10, and is adapted for expression by at least one promoteroperably linked to said nucleotide sequence such that both sense andantisense molecules are transcribed from said cassette.
 25. The nucleicacid molecule according to claim 24, wherein said cassette is adaptedsuch that both sense and antisense ribonucleic acid molecules aretranscribed from said cassette, and wherein said sense and antisensenucleic acid molecules are adapted to anneal over at least part or allof their length to form a inhibitory RNA or short hairpin RNA.
 26. Thenucleic acid molecule according to claim 24, wherein said nucleic acidmolecule is part of a vector adapted for expression in a plant cell. 27.A plant cell transfected with the nucleic acid molecule of claim 24,wherein said cell has reduced expression of at least one of thepolypeptides.
 28. A process for modifying one or more opiate alkaloids,comprising: i) providing a transgenic plant cell according to claim 19;ii) cultivating said plant cell to produce a transgenic plant; andoptionally iii) harvesting said transgenic plant, or part thereof. 29.The process according to claim 28 wherein said harvested plant or partthereof is dried and opiate alkaloid is extracted.
 30. A process formodifying one or more opiate alkaloids or opiate alkaloid intermediatemetabolites, comprising: i) providing the transgenic microbial cellaccording to claim 23 in culture with at least one opiate alkaloid oropiate alkaloid intermediate metabolite; ii) cultivating the microbialcell under conditions that modify one or more opiate alkaloid or opiatealkaloid intermediate; and optionally iii) isolating said opiatealkaloid or opiate alkaloid intermediate from the microbial cell or cellculture.
 31. The process according to claim 30 wherein said microbialcell is a bacterial cell or fungal/yeast cell.
 32. (canceled)
 33. Amethod to produce a P. somniferum plant that has altered expression of apolypeptide comprising: i) mutagenizing a wild-type seed from a P.somniferum plant that does express said polypeptide; ii) cultivating theseed in i) to produce first and subsequent generations of plants; iii)obtaining seed from the first generation plant and subsequentgenerations of plants; iv) determining if the seed from said first andsubsequent generations of plants has altered nucleotide sequence and/oraltered expression of said polypeptide; v) obtaining a sample andanalysing the nucleic acid sequence of a nucleic acid molecule selectedfrom the group consisting of: a) a nucleic acid molecule comprising anucleotide sequence as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or10; and b) a nucleic acid molecule that hybridises to the nucleic acidmolecule in a) under stringent hybridisation conditions and that encodesa polypeptide with opiate alkaloid biosynthetic activity; and optionallyvi) comparing the nucleotide sequence of the nucleic acid molecule insaid sample to a nucleotide sequence of a nucleic acid molecule of theoriginal wild-type plant.
 34. The method according to claim 33 whereinsaid nucleic acid molecule is analysed by a method comprising: i)extracting nucleic acid from said mutated plants; ii) amplifying a partof said nucleic acid molecule by a polymerase chain reaction; iii)forming a preparation comprising the amplified nucleic acid and nucleicacid extracted from wild-type seed to form heteroduplex nucleic acid;iv) incubating said preparation with a single stranded nuclease thatcuts at a region of heteroduplex nucleic acid to identify a mismatch insaid heteroduplex; and v) determining the site of the mismatch in saidnucleic acid heteroduplex.
 35. The method according to claim 33, whereinsaid P. somniferum plant has enhanced opiate alkaloid biosyntheticactivity.
 36. The method according to claim 33, wherein said P.somniferum plant has reduced or abrogated opiate alkaloid biosyntheticactivity.
 37. A P. somniferum plant obtained by the method of claim 33.38. A P. somniferum plant comprising a viral vector that includes all orpart of a gene comprising the nucleic acid molecule according toclaim
 1. 39. The plant according to claim 38 wherein said gene or partis encoded by a nucleic acid molecule selected from the group consistingof: i) a nucleic acid molecule comprising a nucleotide sequence asrepresented in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and ii) anucleic acid molecule comprising a nucleotide sequence that hybridisesunder stringent hybridisation conditions to a nucleic acid molecule in(i) and which encodes a polypeptide with opiate alkaloid biosyntheticactivity.
 40. The plant according to claim 39 wherein said nucleic acidmolecule comprises a nucleotide sequence selected from the groupconsisting of: SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28, 29 and
 30. 41.A viral vector comprising all or part of the nucleic acid molecule ofclaim
 1. 42. (canceled)
 43. An isolated polypeptide selected from thegroup consisting of: i) a polypeptide comprising the amino acid sequenceas shown in SEQ ID NO: 17; ii) a polypeptide modified by additiondeletion or substitution of at least one amino acid residue of thesequence presented in SEQ ID NO: 17 and which has retained or enhancedcytochrome P450 activity; iii) a polypeptide comprising the amino acidsequence as shown in SEQ ID NO:18; iv) a polypeptide modified byaddition, deletion or substitution of at least one amino acid residue ofthe sequence presented in SEQ ID NO: 18 and which has retained orenhanced carboxylesterase activity; v) a polypeptide comprising theamino acid sequence as shown in SEQ ID NO: 19; vi) a polypeptidemodified by addition, deletion or substitution of at least one aminoacid residue of the sequence presented in SEQ ID NO: 19 and which hasretained or enhanced short-chain dehydrogenase/reductase activity; vii)a polypeptide comprising-an amino acid sequence as shown in SEQ ID NO:20; and viii) a polypeptide modified by addition, deletion orsubstitution of at least one amino acid residue of the sequencepresented in SEQ ID NO: 20 and which has retained or enhancedacetyltransferase activity.
 44. The polypeptide according to claim 43,wherein said polypeptide comprises: an amino acid sequence that is atleast 55% identical to the full length amino acid sequence shown in SEQID NO: 17 and which has cytochrome P450 activity; an amino acid sequencethat is at least 46% identical to the full length amino acid sequence inSEQ ID NO: 18 and has carboxylesterase activity; an amino acid sequencethat is at least 47% identical to the full length amino acid sequence inSEQ ID NO: 19 and which has short-chain dehydrogenase/reductaseactivity; or an amino acid sequence that is at least 67% identical tothe full length amino acid sequence in SEQ ID NO: 20 and which hasacetyltransferase activity. 45-50. (canceled)
 51. An isolated nucleicacid molecule that encodes a gene cluster of one or more polypeptidesinvolved in the biosynthesis of opiate alkaloids or intermediatescomprising or consisting of a nucleotide sequence selected from thegroup consisting of: i) a nucleotide sequence as represented by thesequence shown in SEQ ID NO: 7, 8, 9 or 10; ii) a nucleotide sequence,wherein said sequence is degenerate as a result of the genetic code tothe nucleotide sequence defined in (i); iii) a nucleic acid molecule,the complementary strand of which hybridizes under stringenthybridization conditions to the sequence in SEQ ID NO: 7, 8, 9 or 10wherein said nucleic acid molecule encodes a polypeptide involved in thebiosynthesis of P. somniferum opiate alkaloids or intermediates in thebiosynthesis of opiate alkaloids; iv) a nucleotide sequence that encodesa polypeptide comprising an amino acid sequence as represented in SEQ IDNO: 17, 18, 19 or 20; and v) a nucleotide sequence that encodes apolypeptide modified by addition, deletion or substitution of at leastone amino acid residue as represented in iv) above and which hasretained or enhanced opiate alkaloid biosynthetic activity.